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Setdb1 is required for myogenic differentiation of C2C12 myoblast cells via maintenance of MyoD expression.

Song YJ, Choi JH, Lee H - Mol. Cells (2015)

Bottom Line: Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification.We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on.Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, College of Natural Science, Inha University, Incheon 402-751, Korea.

ABSTRACT
Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification. Functions of Setdb1 during development have been extensively studied in embryonic and mesenchymal stem cells as well as neurogenic progenitor cells. But the role of Sedtdb1 in myogenic differentiation remains unknown. In this study, we report that Setdb1 is required for myogenic potential of C2C12 myoblast cells through maintaining the expressions of MyoD and muscle-specific genes. We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on. Gene expression profiling analysis using oligonucleotide micro-array and RNA-Seq technologies demonstrated that depletion of Setdb1 results in downregulation of MyoD as well as the components of muscle fiber in proliferating C2C12 cells. In addition, exogenous expression of MyoD reversed transcriptional repression of MyoD promoter-driven lucif-erase reporter by Setdb1 shRNA and rescued myogenic differentiation of C2C12 myoblast cells depleted of endogenous Setdb1. Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

No MeSH data available.


Related in: MedlinePlus

Exogenous MyoD can restore myogenic potential in Setdb1-depleted C2C12 myoblast cells. (A) Exogenous MyoD can transactivate MyoD-luciferase reporter in C2C12 cells depleted of Setdb1. C2C12 cells stably expressing Setdb1 shRNA or control vector were transfected with plasmid expressing myc-MyoD together with a MyoD-luciferase reporter. The empty vector (pcDNA3) was added to adjust the total amount of transfected DNA to 1.0 μg. Data are presented as relative luciferase activity to the control (empty vector) and expression of exogenous Myc-MyoD was verified by Western blot; Data shown are representative of three independent experiments performed in triplicate, and error bars indicate standard deviation. (B) Proliferating C2C12 myoblast cells stably expressing Setdb1 shRNA or control C2C12 cells were infected with retroviruses expressing MyoD. Empty vector (pLZRS-IRES-GFP) was used as a control. Cells were harvested prior to and 72 hours after induction of differentiation and total proteins were extracted. Differentiation was assessed by western blot analysis using antibody against MHC, and exogenous expression of MyoD was shown to confirm retroviral infection (C) Setdb1 is required for myogenic potential via maintenance of endogenous MyoD expression. In our proposed model, we speculate that Setdb1 inhibits repressor (X) of MyoD that could compete with MyoD in normal proliferating C2C12 myoblast cells. However, depletion of Setdb1 leads to inhibition of differentiation as this potential repressor is relieved from suppression.
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f6-molce-38-4-362: Exogenous MyoD can restore myogenic potential in Setdb1-depleted C2C12 myoblast cells. (A) Exogenous MyoD can transactivate MyoD-luciferase reporter in C2C12 cells depleted of Setdb1. C2C12 cells stably expressing Setdb1 shRNA or control vector were transfected with plasmid expressing myc-MyoD together with a MyoD-luciferase reporter. The empty vector (pcDNA3) was added to adjust the total amount of transfected DNA to 1.0 μg. Data are presented as relative luciferase activity to the control (empty vector) and expression of exogenous Myc-MyoD was verified by Western blot; Data shown are representative of three independent experiments performed in triplicate, and error bars indicate standard deviation. (B) Proliferating C2C12 myoblast cells stably expressing Setdb1 shRNA or control C2C12 cells were infected with retroviruses expressing MyoD. Empty vector (pLZRS-IRES-GFP) was used as a control. Cells were harvested prior to and 72 hours after induction of differentiation and total proteins were extracted. Differentiation was assessed by western blot analysis using antibody against MHC, and exogenous expression of MyoD was shown to confirm retroviral infection (C) Setdb1 is required for myogenic potential via maintenance of endogenous MyoD expression. In our proposed model, we speculate that Setdb1 inhibits repressor (X) of MyoD that could compete with MyoD in normal proliferating C2C12 myoblast cells. However, depletion of Setdb1 leads to inhibition of differentiation as this potential repressor is relieved from suppression.

Mentions: If Setdb1 indirectly regulates endogenous expression of MyoD, it is possible that exogenous expression of MyoD independent of the MyoD promoter could restore the myogenic potential in Setdb1-depleted C2C12 myoblast cells. To test this possibility, we first examined the effect of exogenous MyoD on the MyoD-luciferase reporter in Setdb1-knockdowned C2C12 myoblast cells (Fig. 6A). Consistent with our previous result (Fig. 4A), MyoD-luciferase activity in Setdb1-depleted myoblast cells was consistently lower than that in control C2C12 cells. Interestingly, exogenous expression of MyoD can still transactivate expression of MyoD-luciferase reporter. Finally, we found that retroviral infection of exogenous MyoD resulted in fully differentiated myotubes from Setdb1-depleted C2C12 myoblast cells. This result strongly suggests that Setdb1 plays an important role in maintenance of endogenous MyoD, the perturbation of which could lead to deregulated differentiation (Fig. 6B and Supplementary Fig. S3).


Setdb1 is required for myogenic differentiation of C2C12 myoblast cells via maintenance of MyoD expression.

Song YJ, Choi JH, Lee H - Mol. Cells (2015)

Exogenous MyoD can restore myogenic potential in Setdb1-depleted C2C12 myoblast cells. (A) Exogenous MyoD can transactivate MyoD-luciferase reporter in C2C12 cells depleted of Setdb1. C2C12 cells stably expressing Setdb1 shRNA or control vector were transfected with plasmid expressing myc-MyoD together with a MyoD-luciferase reporter. The empty vector (pcDNA3) was added to adjust the total amount of transfected DNA to 1.0 μg. Data are presented as relative luciferase activity to the control (empty vector) and expression of exogenous Myc-MyoD was verified by Western blot; Data shown are representative of three independent experiments performed in triplicate, and error bars indicate standard deviation. (B) Proliferating C2C12 myoblast cells stably expressing Setdb1 shRNA or control C2C12 cells were infected with retroviruses expressing MyoD. Empty vector (pLZRS-IRES-GFP) was used as a control. Cells were harvested prior to and 72 hours after induction of differentiation and total proteins were extracted. Differentiation was assessed by western blot analysis using antibody against MHC, and exogenous expression of MyoD was shown to confirm retroviral infection (C) Setdb1 is required for myogenic potential via maintenance of endogenous MyoD expression. In our proposed model, we speculate that Setdb1 inhibits repressor (X) of MyoD that could compete with MyoD in normal proliferating C2C12 myoblast cells. However, depletion of Setdb1 leads to inhibition of differentiation as this potential repressor is relieved from suppression.
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f6-molce-38-4-362: Exogenous MyoD can restore myogenic potential in Setdb1-depleted C2C12 myoblast cells. (A) Exogenous MyoD can transactivate MyoD-luciferase reporter in C2C12 cells depleted of Setdb1. C2C12 cells stably expressing Setdb1 shRNA or control vector were transfected with plasmid expressing myc-MyoD together with a MyoD-luciferase reporter. The empty vector (pcDNA3) was added to adjust the total amount of transfected DNA to 1.0 μg. Data are presented as relative luciferase activity to the control (empty vector) and expression of exogenous Myc-MyoD was verified by Western blot; Data shown are representative of three independent experiments performed in triplicate, and error bars indicate standard deviation. (B) Proliferating C2C12 myoblast cells stably expressing Setdb1 shRNA or control C2C12 cells were infected with retroviruses expressing MyoD. Empty vector (pLZRS-IRES-GFP) was used as a control. Cells were harvested prior to and 72 hours after induction of differentiation and total proteins were extracted. Differentiation was assessed by western blot analysis using antibody against MHC, and exogenous expression of MyoD was shown to confirm retroviral infection (C) Setdb1 is required for myogenic potential via maintenance of endogenous MyoD expression. In our proposed model, we speculate that Setdb1 inhibits repressor (X) of MyoD that could compete with MyoD in normal proliferating C2C12 myoblast cells. However, depletion of Setdb1 leads to inhibition of differentiation as this potential repressor is relieved from suppression.
Mentions: If Setdb1 indirectly regulates endogenous expression of MyoD, it is possible that exogenous expression of MyoD independent of the MyoD promoter could restore the myogenic potential in Setdb1-depleted C2C12 myoblast cells. To test this possibility, we first examined the effect of exogenous MyoD on the MyoD-luciferase reporter in Setdb1-knockdowned C2C12 myoblast cells (Fig. 6A). Consistent with our previous result (Fig. 4A), MyoD-luciferase activity in Setdb1-depleted myoblast cells was consistently lower than that in control C2C12 cells. Interestingly, exogenous expression of MyoD can still transactivate expression of MyoD-luciferase reporter. Finally, we found that retroviral infection of exogenous MyoD resulted in fully differentiated myotubes from Setdb1-depleted C2C12 myoblast cells. This result strongly suggests that Setdb1 plays an important role in maintenance of endogenous MyoD, the perturbation of which could lead to deregulated differentiation (Fig. 6B and Supplementary Fig. S3).

Bottom Line: Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification.We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on.Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, College of Natural Science, Inha University, Incheon 402-751, Korea.

ABSTRACT
Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification. Functions of Setdb1 during development have been extensively studied in embryonic and mesenchymal stem cells as well as neurogenic progenitor cells. But the role of Sedtdb1 in myogenic differentiation remains unknown. In this study, we report that Setdb1 is required for myogenic potential of C2C12 myoblast cells through maintaining the expressions of MyoD and muscle-specific genes. We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on. Gene expression profiling analysis using oligonucleotide micro-array and RNA-Seq technologies demonstrated that depletion of Setdb1 results in downregulation of MyoD as well as the components of muscle fiber in proliferating C2C12 cells. In addition, exogenous expression of MyoD reversed transcriptional repression of MyoD promoter-driven lucif-erase reporter by Setdb1 shRNA and rescued myogenic differentiation of C2C12 myoblast cells depleted of endogenous Setdb1. Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

No MeSH data available.


Related in: MedlinePlus